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Our favorite BioBricks

No. 1: BBa_K1150020 - uniCAS Activator

Our uniCAS Activator

No. 2: BBa_K1150024 - uniCAS Histone Modifier

Figure X: CMV:dCas9-G9a (BBa_K1150024) dCas9 was fused via a 3 amino acid linker to G9a. The resulting fusion protein was flanked by NLS sequences and tagged by a HA epitope. The CMV promoter and BGH terminator were chosen to control gene expression.

This device combines the dCas9 protein with the SET-domain of the murine histone methyltransferase G9a. dCas9 enables not only sequence specific, but also multiple targeting of any requested DNA sequence. Hence, coupling of dCas9 to the effector G9a allows for specific methylation of histone (Figure XXXX).
Such methylations are a hallmark of gene repression. One interesting fact about histone modification is the capability to spread the activity state over the surrounding chromatin via reader proteins. So the information of e.g. "repressed state" can, once specifically introduced, be propagated over a whole locus.

We included the G9a histone methylase to the uniCAS toolkit for specific histone methylation. With the device BBa_K1150024, consisting of dCas9 and G9a, we were able to decrease the endogenous VEGF expression in HEK293T cells about 50%, depending on the locus targeted (Figure XXXXXX).

Chromatin remodeling, resulting in repression of endogenous genes, is possible by fusing the histone methyltransferase G9a to dCas9.
dCas9-G9a is our most efficient repressiv device!

Figure X: Endogenous, stable repression by dCas9-G9a HEK293T cells have been trancfected with the BBa_K1150024 device and the RNAimer plasmid containing the different crRNA target sites for the endogenous VEGF locus. 12 hours after transfection the medium was change and 24 hours after medium change we harvested the supernatant and performed VEGF measurments by ELISA. As a control that the repressive effect of our proteins is not based on the sterical block of the transcription, we tested against the catalytic inactive dCas9. So every detectable effect is due to the G9a targeted to this locus.(n=3, p<0.05 is marked by asterisks)

No. 3: BBa_K1150034 - uniCAS RNAimer

Fig. 1: RNAimer (BBa_K1150034) Our RNA plasmid contains the tracrRNA and a site where the desired crRNA can be inserted. Both RNAs are driven by different human RNA polymerase III promoters, H1 and U6. The combination of multiple crRNAs can be easily done by digestion with enzymes of prefix and suffix and ligation of these parts and the RNAimer backbone according to iGEM standard assembly.